Laser desorption ionization (LDI) is a technique in which laser light is delivered onto a sample to help the transfer of electrons within the substance that has absorbed the laser light. Matrix-assisted laser desorption ionization (MALDI) is a technique suitable for an analysis of samples that barely absorb laser light or samples that will be easily damaged by laser light, such as protein. In this technique, a substance that is highly absorptive of laser light and easy to ionize is mixed beforehand into the sample, and this mixture is irradiated with laser light to ionize the sample. Particularly, mass spectrometers using the MALDI technique can analyze high molecular compounds having large molecular weights without severely dissociating them. Moreover, mass spectrometers of this type are suitable for microanalysis. Due to these characteristics, the MALDI mass spectrometers are widely used in biosciences and other fields. In the following description, mass spectrometers with an ion source using an LDI or MALDI technique are generally referred to as the
FIG. 5 is a schematic view of a conventional LDI-MALDI-MS having a typical construction. This system includes a vacuum chamber 10, which is evacuated by a vacuum pump (not shown). The chamber 10 contains a stage 13, ion transport optical system 16, mass analyzer 17, detector 18 and other components arranged in an approximately straight line. Located outside the chamber 10 are a laser-delivering unit 20, laser-condensing optical system 22, CCD camera 23, observation optical system 24 and other components. A sample to be analyzed 15 is applied or placed on a sample plate 14. This plate 14 is set on a stage 13, which can be horizontally moved along the x and y directions. Examples of the ion transport optical system 16 include an electrostatically-operated electromagnetic lens, a multi-polar radio-frequency ion guide, or a combination of these devices. The mass analyzer 17 may be a quadrupole mass spectrometer, ion trap, time-of-flight mass spectrometer, magnetic sector mass spectrometer, or other types of mass spectrometers.
An analysis with the previous mass spectrometer involves the following steps: An operator initially determines which portion of the sample 15 should be analyzed. To help him/her with this task, an image of the sample 15 is captured with the CCD camera 23 through the observation window 12 and the observation optical system 24, which are located in a side of the vacuum chamber 10, and the image is displayed on a monitor (not shown). FIGS. 9(a) and 9(b) are top views of the stage 13.
In FIGS. 9(a) and 9(b), the rectangle 23a indicated by the dotted line corresponds to the scope of the CCD camera 23a, and the approximately spherical, shaded range 21a corresponds to the irradiation range of the laser light 21. The scope 23a is larger than the convergence diameter of the laser light 21. The center of the irradiation range 21a of the laser light 21 approximately coincides with that of the scope 23a. Accordingly, the irradiation range 21a of the laser light 21 can be completely covered by the scope 23a, as shown in FIG. 9(a). The diameter of the converged laser light 21 is generally smaller than the sample 15, also as shown in FIG. 9(a).
Observing the image of the sample 15 within the scope 23a, the operator appropriately moves the stage 13 along the x and y axes to locate a target portion to be analyzed. In FIGS. 9(a) and 9(b), for example, the target portion is indicated by the point 15a. Then, he/she brings the target portion 15a to the center of the laser irradiation range 21a, as shown in FIG. 9(b).
Subsequently, the operator gives a command for starting the analysis, upon which the laser-delivering unit 20 starts emitting the laser light 21. This light is condensed by the laser-condensing optical system 22 and then delivered through the irradiation window 11, which is located in a side wall of the vacuum chamber 10, onto a point in the vicinity of the target portion 15a on the sample 15. The laser light 21 thus delivered ionizes various substances contained in the sample 15. The ions thereby produced are emitted vertically, i.e. in directions approximately perpendicular to the sample plate 14. These ions are converged by the ion transport optical system 16 into the mass analyzer 17. The mass analyzer 17 separates the ions according to their mass-to-charge ratios and sends them to the detector 18. The detector 18 produces an electric current indicative of the number of the received ions and outputs the electric current as a detection signal. The mass analyzer 17 can be operated so that it scans a specific range of mass-to-charge ratios. In this case, with the lapse of time, the detector 18 consecutively detects several kinds of ions having different mass-to-charge ratios. The detection signals thereby produced can be used to create a mass spectrum with a data processor (not shown).
In the previous construction, the CCD camera 23 for capturing an image and displaying it on the monitor can be replaced by an eyepiece for enabling the operator to visually and directly observe a microscopic image of the sample. The observation optical system 24 may have various constructions depending on the spatial resolution for observation and/or the operational distance; it may be comprised of a single element, a module of multiple elements combined, or even a larger unit including a plurality of such modules. The laser-condensing optical system 22 may have various constructions depending on the specifications of the laser-delivering unit 20 and/or the requirement for the diameter of conversion; as in the case of the observation optical system 24, it may be comprised of a single element, a module of multiple elements combined, or even a larger unit including a plurality of such modules.
Improving the spatial resolution of the LDI/MALDI-MS will enable advantageous applications of the apparatus. For example, it can be used for examining body tissue to analyze the cause and process of a disease, clarify vital functions, or acquire versatile knowledge about sample preparation. However, conventional types of LDI/MALDI-MSs on the market are far from being available for such purposes since the diameter of converged laser light is too large (e.g. several hundreds of micrometers) and the scope of the CCD camera (or eyepiece) is too large (e.g. several millimeters in length or width). As a conventional example, Non-Patent Document 1 discloses an analysis method in which the laser light is converged to a level of several tens of micrometers in diameter. However, this level of convergence diameter is not sufficient for examining a specific portion of a living cell since the cell itself is as small as several tens of micrometers. Accordingly, it is preferably necessary to achieve a high spatial resolution of approximately a few to several micrometers.
To improve the spatial resolution of an analysis by LDI/MALDI-MS, it is necessary to:    (1) improve the spatial resolution for observing the sample;    (2) reduce the diameter of the converged laser light, which is to be delivered onto the sample;    (3) project the laser light accurately at the target point on the sample; and    (4) design the irradiation/observation optical systems so that they do not deteriorate the ion-detecting efficiency.
Some of the conventional mass spectrometers include special improvements for achieving higher spatial resolutions. For example, FIG. 6 is a schematic view of the construction of a mass spectrometer disclosed in Non-Patent Document 2. The components that are identical or equivalent to those shown in FIG. 5 are indicated by the same numerals. In the present mass spectrometer, the observation optical system 24 in FIG. 5 is replaced by a zoom lens 26, and an aperture 25 for limiting the passage area of light is provided in the vicinity of the aperture of the laser-delivering unit 20.
In FIG. 5, the laser light 21 is assumed to turn to a parallel beam immediately after it is emitted from the laser-delivering unit 20. However, strictly speaking, this is not always true; in many cases, the beam minimizes its diameter at a point within the laser-delivering unit 20 or immediately after leaving the unit. After passing that point, the beam gradually increases its diameter with its travel. In the case where the light is an ideal parallel beam, if the passage area of the light is limited by the aperture 25 as shown in FIG. 6, the numerical aperture of the laser-condensing optical system will decrease. Therefore, the convergence diameter of the laser light will increase rather than decrease. By contrast, in the case where the light is a diverging beam, the aperture 25 will reduce the minimum diameter of the beam, so that the convergence diameter, which reflects the aforementioned minimum diameter, will decrease. Unfortunately, the aperture 25 blocks a portion of the light and thereby lowers the power of the laser light. This problem can be avoided by replacing the aperture 25 with a lens for pre-focusing the light.
However, in any cases, the construction shown in FIG. 6 has a problem in that the numerical aperture of the optical systems is small since both the laser-condensing optical systems 22 and the observation optical system have large working distances. Therefore, in terms of the conversion diameter of the laser light 21 and the spatial resolution for observation, this construction cannot significantly exceed the other conventional ones.
One idea for reducing the working distance of the laser-delivering optical system and observation optical system is to place the optical systems 22 and 24 closer to the sample 15, as shown in FIG. 7. This arrangement increases the numerical apertures of the two optical systems 22 and 24, whereby the spatial resolution for observation is improved and the convergence diameter of the laser light 21 is reduced. In this arrangement, it is necessary to leave the space around the axis C as widely open as possible since the ions generated at the irradiated portion of the sample 15 are given the kinetic energy in directions approximately parallel to the surface normal to the sample plate 14, or along the axis C, and begin to fly in those directions; these ions must be prevented from being lost due to the collision with the observation optical system 24 and the laser-condensing optical system 22. Furthermore, the optical systems must be arranged so that one optical system does not interfere with any element or optical axis of the other optical system. Due to these restrictions, there is a limit for the optical systems 22 and 24 to come closer to the sample 15.
This limitation particularly causes a problem for the observation optical system 24. For example, an ultraviolet laser light can be easily converged to a diameter of a few micrometers with a working distance of several tens of millimeters by using a common, inexpensive condensing lens as the laser-condensing optical system 22. To prevent interference between the two optical systems, it is desirable that the observation optical system 24 should also have an approximately equal working distance. As another requirement, the observation optical system should have a resolution comparable to the convergence diameter of the laser light in order to assuredly move a micro-sized target portion of the sample within the irradiation range of the laser light. However, since the observation optical system does not use the highly coherent laser light but normal visible light, it is almost impossible to achieve a spatial resolution of a few micrometers with a working distance of several tens of millimeters. Thus, in the construction shown in FIG. 7, although the convergence diameter of the laser light can be reduced to a desired level, it is difficult to improve the spatial resolution for observation to a level comparable to the convergence diameter.
Non-Patent Document 3 discloses a mass spectrometer constructed as shown in FIG. 8. This construction includes a perforated optical system and a perforated mirror 28, both located above the stage 13, and a wavelength selection mirror 29 located outside the observation window 12. The perforated optical system is commonly used for both observation and laser condensation. An image of the sample 15 is captured with the CCD camera 23 through the optical system 27, the perforated mirror 28, the observation window 12 and the wavelength selection mirror 29. The laser light 21 emitted from the laser-delivering unit 20 passes through the wavelength selection mirror 29 and the observation window 29. Then, it is reflected downwards by the perforated mirror 28, condensed by the perforated optical system 27 onto the sample 15. The irradiation of laser light generates ions from the sample 15. These ions pass through the perforations of the perforated optical system 27 and the perforated mirror 28 and then reach the ion transport optical system 16.
In this construction, the perforated optical system 27 can be placed adequately close to the sample 15 without causing the previously stated problems, such as the interference of the optical systems. Therefore, the spatial resolution for observation can be significantly improved and the convergence diameter of the laser light can be considerably reduced. However, even through the ions begin to fly in directions approximately parallel to the surface normal to the sample plate 14, these ions also have velocity components in the direction perpendicular to the surface normal, so that some of the ions will be blocked by the perforated optical system 27 or the perforated mirror 28. This will inevitably lower the ion transport efficiency. Another problem exists in that the ion-generating efficiency is lower than that of the previous constructions shown in FIG. 5 or other figures since the laser light 21 loses its energy every time it passes through or reflected by the wavelength selection mirror 29, the perforated optically system 27, the perforated mirror 28 and other components.
[Non-Patent Document 1] P. Chaurand et al., “Profiling and imaging proteins in tissue sections by MS”, Analytical Chemistry, 2004, Vol.76, No.5, p.86A-93A
[Non-Patent Document 2] R. M. Caprioli et al., “Molecular imaging of biological samples: Localization of peptides and proteins using MALDI-TOF MS”, Analytical Chemistry, 1997, Vol. 69, No. 23, p.4751-4760
[Non-Patent Document 3] B. Spengler et al., “Scanning Microprobe Matrix-Assisted Laser Desorption Ionization (SMALDI) Mass Spectrometry: Instrumentation for Sub-Micrometer Resolved LDI and MALDI Surface Analysis,” Journal of American Society for Mass Spectrometry, 2002, Vol.13, No.6, p.735-748